NFATc1 induction by an intronic enhancer restricts NKT γδ cell formation

Summary In thymus, the ablation of T cell receptor (TCR)-activated transcription factor NFATc1 or its inducible isoforms during the double-negative (DN) stages of thymocyte development leads to a marked increase in γδ thymocytes whereas the development of αβ thymocytes remains mostly unaffected. These γδ thymocytes are characterized by the upregulation of the promyelocytic leukemia zinc-finger factor (PLZF), the “master regulator” of natural killer T (NKT) cell development, and the acquisition of an NKT γδ cell phenotype with higher cell survival rates. The suppressive function of NFATc1 in NKT γδ cell formation critically depends on the remote enhancer E2, which is essential for the inducible expression of NFATc1 directed by its distal promoter P1. Thus, the enhancer deciphers a strong γδ TCR signal into the expression of inducible NFATc1 isoforms resulting in high levels of NFATc1 protein that are essential to control the numbers of NKT γδ cells.


INTRODUCTION
The thymus is a primary lymphoid organ and the site where lymphoid progenitors, moving from fetal liver or bone marrow, differentiate into T cells. In adult mice, common lymphoid progenitors (CLPs) from the bone marrow (BM) enter the thymus from the blood at the cortico-medullary junction, migrate into the organ, and differentiate. Along with conventional ab T cells, so-called unconventional T cells including gd T cells, invariant (iNKT) and diverse natural killer T (NKT) cells (dNKT), NKT gd cells, and the CD8aa + TCRab intraepithelial lymphocyte precursors (IELPs) develop in the thymus. 1,2 Thymocyte development can be classified into different stages based on the expression of surface markers, including the co-receptors CD4 and CD8, and the corresponding T cell receptors (TCRs). During the first stages of T cell lineage development, thymocytes lack both CD4 and CD8 co-receptors expression and are therefore named double-negative (DN). Based on the surface expression of CD44 and CD25, DN thymocytes can be subdivided into four distinct subpopulations: ETP-DN1 (CD44 + CD25 À ), DN2 (CD44 + CD25 + ), DN3 (CD44 À CD25 + ), and DN4 (CD44 À CD25 À ) cells. 3,4 At the point of transition from DN2 to DN3 stages, expression of the Rag1 and Rag2 genes (recombination activating genes 1 and 2) occurs. Tcrg, Tcrd, and Tcrb loci are rearranged in thymocytes thus leading to the expression of the TCRgd or the b-chain of the TCR. 4 The TCRb chain pairs with a surrogate a chain (pre-Ta) to form the pre-T cell receptor (pre-TCR). This step represents the time point of ab and gd bifurcation. 5 gd T cells are a unique and well-conserved population of lymphoid cells. Different from ab T and B cells, gd T cells display characteristics of cells of both the innate and adaptive immune system. In mice, they account for 4% of T cells in the thymus and secondary lymphoid organs. It is still controversial which signals induce the common DN progenitors to follow one or the other lineage. Current studies support the TCRdependent strength model. Similarly, it has been shown that NKT cells, a population belonging to the unconventional innate-like group, require a strong TCR signal for selection. [6][7][8] NKT cells are a subset of innate-like T cells, which share innate characteristics of NK cells and adaptive functions like T lymphocytes, therefore owning the ability to bridge innate and adaptive immunity. 9 They undergo a selection in the thymus and recognize various lipid antigens presented on CD1d molecules. 10 In this study, we investigated the role of NFATc1 during the DN stages of early thymocyte development further. While in thymi of both Rag1Cre-Nfatc1 fl/fl and Rag1Cre-E2 fl/fl mice, in which the synthesis of all or inducible NFATc1 proteins is abolished, a moderate decrease in overall thymocyte numbers was observed, we detected a marked expansion in gd thymocytes. Those gd thymocytes exhibited a strong increase in the expression of PLZF, the ''master regulator'' of NKT cells, and the acquisition of an NKT gd phenotype. These data suggest that, due to the induction by gd TCR activation in DN3 thymocytes, high NFATc1 levels increase the susceptibility for apoptosis in NKT gd cells and thereby control their expansion.

Lack of NFATc1 activity leads to an increased number of gd thymocytes
In our previous study, ablation of NFATc1 in hematopoietic T cell progenitors led to the shrinking of lymphatic organs, a dramatic decrease in thymic cellularity, and a block of thymocyte development at the DN1 stage. 30 To check whether the inactivation of the Nfatc1 gene in early DN thymocytes exerts a similarly dramatic effect on overall thymic development, we investigated thymocyte development in Rag1Cre-Nfatc1 fl/fl mice. In those mice, the Cre recombinase expressed under the control of the Rag1 locus causes the inactivation of the target gene very efficiently at the DN2 to DN3 stages. 31 This resulted in a strong decrease in Nfatc1 RNA levels both in DN and in DP and SP thymocytes ( Figure S1A). However, the loss of NFATc1 expression in early thymocytes resulted only in a mild reduction of overall thymic cellularity in Rag1Cre-Nfatc1 fl/fl mice ( Figure 1A). While a subtle decrease in DP and CD4 + single-positive thymocytes was detected, almost no difference was observed in the distribution of subpopulations of DN thymocytes between Nfatc1 fl/fl control and Rag1Cre-Nfatc1 fl/fl mice ( Figures 1B and 1C). When we studied the lineage distribution of thymocytes from Nfatc1 fl/fl , Rag1Cre-Nfatc1 fl/+ , and Rag1Cre-Nfatc1 fl/fl mice by flow cytometry, a slight but non-significant decrease was observed in the percentage and absolute numbers of TCRab thymocytes in total population ( Figures 1D and S1B). However, these assays revealed a conspicuous NFATc1 dose-dependent increase in the percentage of gd thymocytes between those mouse lines. Absolute numbers of gd thymocytes in Rag1Cre-Nfatc1 fl/fl mice increased more than 2-fold compared to control mice ( Figure 1D). These results prompted us to further analyze gd lineage development. We performed intracellular staining of TCRb and TCRd chains. The data of flow cytometry assays revealed again a marked increase in TCRd + cells in the populations of total and DN thymocytes from Rag1Cre-Nfatc1 fl/fl mice, and the proportion of TCRb + cells was comparable both in total and in DN thymocytes. It is Noteworthy that we also observed a significant increase in the population of NFATc1-deficient thymocytes expressing both the intracellular TCRb and TCRd chains (0.18% in total Nfatc1 fl/fl vs 0.35% in Rag1Cre-Nfatc1 fl/fl and 1.48% in ll OPEN ACCESS iScience Article DN Nfatc1 fl/fl vs 3.05% in Rag1Cre-Nfatc1 fl/fl ) (Figures S1C and S1D). We also confirmed by RT-PCR analysis that the defect in Nfatc1 expression was not compensated by changes in Nfatc2 and Nfatc3 expression ( Figure S1E). Taken together, these results suggest that during the DN stages of thymocyte development, NFATc1 plays a relevant role in the regulation of gd T cell development.
To exclude the possibility that gd thymocytes accumulate in the thymus, we analyzed the presence of gd T cells in some homing tissues, such as in the intestine and skin. We observed a heightened percentage of gd T cells among the lymphocytes from lamina propria (LP) and skin of Rag1Cre-Nfatc1 fl/fl mice, but not within the intraepithelial lymphocyte (IEL) population ( Figures 1E and S1F). Therefore, the absence of NFATc1 leads to an increase in gd T cells in thymus and peripheral organs. This suggests that NFATc1 plays a role in the phenotype specification of gd subpopulations during thymocyte development.
The absence of NFATc1 leads to a change in gd thymocyte differentiation We next characterized the gd thymocyte population in Rag1Cre-Nfatc1 fl/fl mice for maturation surface markers and functional identity. Among the surface markers that characterize the developmental stages of gd T cells during thymocyte development, we measured the expression of CD24 and CD73 as indicators for cell maturation and commitment. 14 gd thymocytes move along from more immature CD24 + CD73 À cells to mature CD24 À CD73 + stages, 32 where CD73 expression corresponds to a definitive commitment toward the gd lineage. 33 Flow cytometry showed an increase in the percentage of gd thymocytes with no or lower expression of CD24 and an increase in CD73 surface marker expression on Rag1Cre-Nfatc1 fl/fl gd thymocytes ( Figure 2A). These data suggest that NFATc1-deficient gd thymocytes are fully committed to the gd cell lineage.
Previous research has shown that the expression of CD44 and CD45RB surface markers on gd thymocytes correlates with a functional fate in the thymus. 32 Our analysis of surface markers showed a strong increase in CD44 + CD45RB + cells in gd thymocytes lacking NFATc1 ( Figure 2B).

NFATc1-deficient gd T cells acquire an NKT gd cell phenotype
To check if the ablation of NFATc1 in DN thymocytes leads to an altered differentiation in those gd thymocytes, we performed RNA sequencing (RNA-seq) assays using gd thymocytes isolated from Rag1Cre-Nfatc1 fl/fl and control mice ( Figure 3A). We observed downregulation of the Cd24a gene, which was obvious from flow cytometry already ( Figure 2A). The RNA-seq data also confirmed the upregulation of Il4 gene expression ( Figure 2C). Strikingly, unbiased cell-type identification using the transcriptome from isolated NFATc1-deficient gd thymocytes identified a signature associated with NKT cells ( Figure 3B).
As a part of the NKT population retains the expression of CD4 during development 10,12,34 and several studies showed CD4 expression in an expanded population of NKT gd cells in mouse models with TCR signaling defects, 16,35 we measured CD4 expression on gd thymocytes. Flow cytometry revealed that in Rag1Cre-Nfatc1 fl/fl mice 4-fold more gd thymocytes expressed the CD4 co-receptor compared to those in Nfatc1 fl/fl mice (41.7% Rag1Cre-Nfatc1 fl/fl and 9.05% Nfatc1 fl/fl ) ( Figure 3C). Flow cytometry of the ''classical'' NK1.1 surface marker revealed a significant increase in its expression on NFATc1 À/À gd thymocytes ( Figure 3D). In addition, we observed an increase in CD4 + gd thymocytes expressing Vg1.1 and Vd6.3 gene iScience Article segments (TCRV), typical for NKT gd cells, in mice bearing NFATc1-deficient thymocytes ( Figure 3E). However, analysis using Cd1d tetramers in thymi from Rag1Cre-Nfatc1 fl/fl and control mice revealed no differences in the conventional NKT ab population ( Figure S2A).
Among several differentially transcribed transcription factors, we recognized that Zbtb16, the gene which encodes the transcription factor PLZF, was strongly upregulated in the absence of NFATc1 ( Figure 3A). PLZF has been described as a ''master regulator'' of NKT and NKT gd development. 15,18 We also observed a similar strong upregulation of Zbtb16 mRNA using Rag1Cre-Nfatc1 fl/fl DN thymocytes in RT-PCR assays ( Figure S2B). Flow cytometry showed a much higher number of gd thymocytes from Rag1Cre-Nfatc1 fl/fl mice compared to Nfatc1 fl/fl control mice (47% vs 2.6%) expressing PLZF factor ( Figure 3F). This increase in PLZF + cells is most prominent in the CD4 + gd subpopulation of Rag1Cre-Nfatc1 fl/fl thymocytes (Figure S2C). To prove that CD4 + gd thymocyte expansion is led by an intrinsic mechanism, we analyzed the thymocytes from bone marrow chimeric mice. CD45.1 + Rag2 À/À gc À/À recipient mice were reconstituted with CD45.1 + CD45.2 + wild-type (WT) and CD45.2 + Rag1Cre-Nfatc1 fl/fl bone marrow cells in a ratio of 1:1. While both types contribute equally to the generation of all thymic populations ( Figure S2D), the percentage of PLZF + thymocytes was significantly increased in the NFATc1-deficient compartment of gd thymocytes compared to wild type ( Figure 3G).

Reduced cell death in NFATc1-deficient NKT gd thymocytes
Pathway analysis of our RNA-seq data revealed in Rag1Cre-Nfatc1 fl/fl gd thymocytes a downregulation of genes involved in the positive control of cell death, pointing out a function of NFATc1 in controlling gd thymocyte survival ( Figure 4A). To address the cause of NKT gd thymocyte increase in Rag1Cre-Nfat/c1 fl/fl thymi, we measured the apoptosis levels of different thymocyte subsets by annexin V staining. When PLZF + thymocytes were gated for TCR Vd6.3and Vd6.3 + populations, we observed   Figure S3D). To evaluate the possibility that the lack of NFATc1 could confer a proliferation advantage of Rag1Cre-Nfatc1 fl/fl gd compared to WT thymocytes, we performed a proliferation analysis of gd thymocyte subpopulations by Ki67 protein staining. Our data revealed no significant difference within the TCRVd6.3 + and TCRVd6.3expressing PLZF factor and TCRVg1.1 PLZFand PLZF + subpopulations ( Figures 4B, 4D, S3A, and S3C).
To unravel the molecular mechanisms that support the survival of NKT gd in Nfatc1 À/À thymocytes, we stimulated thymocytes in vitro by aCD3 for 24 h and detected fewer gd thymocytes from Rag1Cre-Nfatc1 fl/fl mice with PLZF expression, compared to untreated cells. The addition of cyclosporin A (CsA), a specific inhibitor of Ca ++ -dependent phosphatase calcineurin, to aCD3-treated cells led to a ''rescue'' in the number of gd thymocytes, which expressed PLZF ( Figure 4E). Taken together, our data show that the ablation of NFATc1 supports the survival of gd thymocytes that express PLZF while strong TCR stimulation in vitro by aCD3 reduces their number.

Strong NFATc1 expression in gd thymocytes
The increase in gd thymocytes upon NFATc1 ablation prompted us to investigate NFATc1 expression in ab and gd thymocytes. Using Nfatc1-eGfp-Bac reporter mice that express an EGFP indicator gene under the control of the Nfatc1 locus, 29,36 we detected similar percentage of gd and ab thymocytes expressing GFP ($93.3%). However, the NFATc1-mediated GFP expression was stronger in gd compared to ab thymocytes, as indicated in the MFI ratio level between the two populations ( Figure 5A). In gated DN thymocytes, we observed a significant difference in NFATc1 expression between gd and ab thymocytes but to a lesser extent compared to that in total thymocytes. ( Figure S4A). Similar results were obtained for gd T cells from lymph nodes and spleen. There, again, NFATc1 was more strongly expressed in gd T cells compared to ab cells ( Figure S4B). The same analysis revealed that the few CD4 + gd thymocytes expressed a higher level of NFATc1 compared to ab thymocytes ( Figure S4C).

Nfatc1 enhancer E2 is essential for the suppression of NKT gd cell development
We showed previously that the expression of pre-TCR in DN3 thymocytes is closely linked to the transcriptional induction of NFATc1 and the generation of NFATc1/a isoforms in these cells. 28 To test whether this NFATc1 induction in DN3 thymocytes suppresses gd lineage development, we investigated thymocyte development in Rag1Cre-E2 fl/fl mice, a newly generated mouse line, in which the remote enhancer E2 28 is deleted ( Figure 5B). This leads to specific inhibition of NFATc1 induction by preventing the expression of NFATc1/a but not of the constitutive NFATc1/b isoforms ( Figures 5C and 5D). As for the complete ablation of all NFATc1 proteins in Rag1Cre-Nfatc1 fl/fl mice, we observed a moderate decrease in the number of total thymocytes ( Figure S5A) but a marked, 7-fold increase in the percentage of gd thymocytes in Rag1Cre-E2 fl/fl mice compared to control littermates (1.17% Rag1Cre-E2 fl/fl vs 0.16% E2 fl/fl control) ( Figure 5E). As iScience Article confirmed by flow cytometry, this increase in gd thymocytes was not paralleled by a significant decrease in ab thymocytes ( Figure S5B).
Similar to Rag1Cre-Nfatc1 fl/fl mice, NFATc1/a-deficient mice showed increased CD24 À CD73 + ( Figure 5F) and CD44 + CD45RB + gd thymocytes as well as an incremented number of gd thymocytes producing IFNg and IL-4 cytokines ( Figures S5C-S5E). The proportion of annexin V-positive gd Rag1Cre-E2 fl/fl thymocytes was similarly reduced as upon total loss of NFATc1 compared to control cells ( Figure S5F). The number of gd thymocytes expressing CD4 co-receptor (8.5% E2 fl/fl and 42% Rag1Cre-E2 fl/fl ) ( Figure 5G) and the Vg1.1 and the Vd6.3 gene segments increased significantly compared to control mice ( Figure 5H). These results indicate that NKT gd cells accumulate in the absence of the enhancer E2 and thereby the induction of NFATc1/a isoforms.

NFATc1 binds to numerous genes expressed in gd cells
To elucidate whether NFATc1 binds prominent genes expressed in gd thymocytes, we investigated the genome-wide binding sites of NFATc1 in thymocytes from transgenic Nfatc1/A-Bio.BirA mice in chromatin immunoprecipitation sequencing (ChIP-seq) assays. In those mice, an additional copy of NFATc1 is expressed from a bacterial artificial chromosome (BAC) transgene, bearing a biotin-tag at the C terminus of NFATc1/A 37,38 of both isoforms NFATc1/a and NFATc1/b. The analysis of ChIP-seq results revealed the common NFAT binding motif ( Figure 6A) within the peaks and allowed us to identify genes with NFATc1 binding sites enclosed by an area of 100 kb around their transcription start site. The Vulcano plot in Figure 6B shows that, compared to Nfatc1 fl/fl , in gd thymocytes from Rag1Cre-Nfatc1 fl/fl mice that are characterized by an expanded population of NKT gd cells, 903 genes are differentially expressed and are a direct target of NFATc1 (indicated by log 10 of MACS2 peaks score, Figure 6B), while 649 genes are not NFATc1 direct targets. Some examples of genes involved in the apoptosis/survival regulation of NKT gd thymocytes are indicated, and the NFATc1 binding is shown in detail for the Cd24a, Casp3, Bmf (Bcl2-modifying factor), and Bcl2a1b genes ( Figure 6C). In addition, the RNA-seq data of thymocytes from Nfatc1/A-Bio.BirA mice expressing this extra copy of NFATc1 revealed that the expression of segments associated with the gd TCRs, such as the Tcrg-V1 gene segment, was downregulated, in contrast to that of ab TCR-associated gene segments ( Figure 6D). These results indicate that overexpressing NFATc1 can suppress gd transcripts.

DISCUSSION
In this study, we investigated the role of the transcription factor NFATc1 during the DN stages of thymocyte development. We show here that the absence of NFATc1 during these early stages of thymocytes affects the phenotypical and functional differentiation of gd T cells. Ablation of NFATc1 in DN thymocytes leads to a marked increase in the number of NKT gd cells that display high levels of the transcription factor PLZF, the ''master regulator'' of NKT cell development, and further characteristics of NKT cells. 15 Using a VavCre-Nfatc1 fl/fl mouse model in which the depletion of NFATc1 occurs in hematopoietic progenitor cells, we observed a complete block of thymocyte development at the DN stages. 30 However, in our study here, Rag1Cre-Nfatc1 fl/fl mice were generated for the specific ablation of NFATc1 starting at DN stages. They exhibited a very moderate decrease in overall thymic cellularity but a marked expansion of gd thymocytes with NKT cell characteristics. Owing to the early and efficient activity of the Rag1Cre system in depleting NFATc1 function shortly ahead of gd lineage choice, we suppose that this effect is cell intrinsic. iScience Article This indicates that NFATc1 does not control the overall thymic T cell development but quite specific steps in the lineage determination of thymocytes.
In thymocytes, the signals delivered by the pre-TCR and TCR complexes differ from those in mature T cells. During development in the thymus, the pre-TCR (composed of a b and a pre-a chains) signals to the cell that the pre-T cell receptor components are properly expressed driving thymocyte survival, proliferation, and acquisition of a ab T cell phenotype. This process is indicated as b-selection. Similarly, at this stage, some thymocytes might express a proper gdTCR licensing them to pass the gd-selection process. Later in development in the thymus, the recognition of major histocompatibility complex molecules displaying a self-antigen by a mature TCR (composed of the b and a chains) signals whether thymocytes are selected positively or negatively. In peripheral mature T cells, in response to an antigen, TCR signaling leads predominantly to the activation of naive or resting T cells. The current ''signal strength theory'' of thymocyte development claims that strong TCR signals, normally induced by a gdTCR, received by DN cells lead to the acquisition of a gd lineage, whereas weaker signals, induced by the pre-TCR, support the generation of ab thymocytes. 39,40 Our data imply that strong signals that induce NFATc1 and its a-isoforms in parallel limit the number of PLZF + CD4 + Vg1.1 + Vd6.3 + thymocyte populations. A similar increase in this population was described for Itk-, Id3-deficient, and SLP-76 mutant mice bearing thymocytes with impaired TCR signal strength. 16,35,41 We hypothesized that the increase of NKT gd population in Rag1cre-Nfatc1 fl/fl mouse reflects the acquisition of a better survival rate upon lack of NFATc1. Our RNA-and ChIP-seq analysis showed that genes involved in the apoptosis pathway, such as Casp3 and Bmf (encoding the proapoptotic BH3-only Bcl-2 modifying factor), 42 are downregulated in NFATc1-deficient gd thymocytes. In addition, the surface marker CD24 is much less expressed on NFATc1-deficient gd thymocytes, compared to control cells. It has been shown that the cross-link of CD24 with a specific antibody drives apoptosis of DN thymocytes. 43 Therefore, in a physiological situation, the induction of those genes by NFATc1 will increase susceptibility to apoptosis. In line with this, our data revealed that the absence of NFATc1 leads to an upregulation of anti-apoptotic members of the Bcl2A1 family (Bcl2a1a, Bcl2a1b, Bcl2a1d). Bcl2A1 was described to enable the survival of DN thymocytes upon a proper pre-TCR signaling. 44 However, a more detailed analysis of Bcl2A1 function by the complete ablation of all Bcl2A1 isoforms revealed no obvious defects in thymocyte development but a decrease in gd T cell numbers in the spleen. 45 On the other hand, a positive correlation between the expression of NFATc1 and Bcl2A1-mediated cell survival has been observed in peripheral lymphocytes, 26,46 suggesting the existence of an alternate mechanism for the survival of thymocytes and peripheral T (and B) cells.
Among the different effector types of gd cells, NKT gd cells share several characteristics with NKT ab cells, such as their surface markers, transcription factors, and cytokine production. PLZF is the ''master regulator'' of several innate-like T cells, including NKT cells. 18 Previous data have shown that PLZF in NKT cells is induced by a strong TCR signals. 17 In our experiments, dampening the intracellular signal transfer from the TCR by inactivation of NFATc1 in the DN thymocytes leads to an increased number of PLZF-expressing gd thymocytes. Reinforcing TCR signaling by stimulation with aCD3 provokes a decrease in PLZF + Nfatc1 À/À deficient gd thymocytes. This decrease could be abolished by CsA, an inhibitor of the Ca 2+ /calcineurin network that controls the activation of NFAT factors. These findings suggest that Ca 2+ /calcineurin/NFAT signals limit the number of NKT gd thymocytes and, thereby, restrict the development of those cells. Future experiments using transgenic gd TCR models aim to underpin this conclusion. 39,47 The transcription factor ThPOK (coded by the Zbtb7b gene) is required to drive immature thymocytes toward the CD4 lineage, 48,49 and it has been shown that PLZF-expressing Vg1.1 + Vd6.3 + T cells also express the CD4 co-receptor. 16 These observations were confirmed in our analysis, which revealed a substantial increase in CD4 + Vg1.1 + Vd6.3 + gd thymocytes. Noteworthy, in our RNA-seq analysis, a marked upregulation of the Zbtb7b gene in Nfatc1 À/À gd thymocytes was observed. In addition, our ChIP-seq assays showed the binding of NFATc1 at the Zbtb7b locus, indicating a direct transcriptional regulation by NFATc1.
The observation that gd thymocytes express higher levels of NFATc1 compared to ab thymocytes led us to assume that, as in peripheral T cells, the induction of NFATc1/a isoforms contributes to the high NFATc1 expression level. 28 Indeed, in the thymus of Rag1Cre-E2 fl/fl mice, in which the deleted enhancer E2 leads to the loss of a isoforms, a similar accumulation of NKT gd was detected. This suggests that gd thymocytes require strong signals for the tight control of NKT gd cell numbers. Taken together, the constitutive expression of NFATc1 in DN thymocytes plays a minor role in their further development, but the induction of NFATc1/a isoforms, regulated by the distal enhancer E2, controls the proper development of NKT gd cells.

Limitations of the study
We have to acknowledge some limitations of our study. We are aware of the importance of further signaling pathways for gd thymocyte development such as the one mediated by the signaling lymphocytic activation molecule (SLAM) receptors. Because the differentiation of thymocytes requires the integration of several signaling pathways together with TCR signaling, this has to be addressed using additional genetic models in the future.

ACKNOWLEDGMENTS
We are particularly thankful to Andreas Rosenwald for his continuous support of this project. We are grateful to Andrew Kueh and Marco Herold for the generation of E2 fl/fl mice. We are also thankful to Maria Grazia Marzella for the critical reading of the manuscript.

DECLARATION OF INTERESTS
The authors declare no competing interests.

Materials availability
The mouse line C57BL/6J Nfatc1 E2 fl/fl generated in this study has been deposited to the Knockout Mouse Project (KOMP), MGI:7428886 (synonym Nfatc1 em1Serf ).

Data and code availability
ChIP-Seq and RNA-Seq data supporting this study are available in the NCBI's Gene Expression Omnibus and are accessible through the GEO series accession number GSE198031. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. This paper does not report the original code.